1. Field of Invention
The invention relates to a colloidal gold sandwich immunoassay method for the identification of antigen or hapten analytes in biological specimens. More particularly, the invention relates to an immunological method for analyzing biological fluids wherein the separation and concentration of solid phase bound colloidal gold labeled binder is conducted on a finite area of a porous film wherein the reaction can be visualized by the human eye.
2. Discussion of Prior Art
Immunodiagnostic testing is rapidly evolving toward more simplistic approaches in the rapid identification and diagnosis of disease states. There is often a need for a simplistic qualitative assay to detect the presence or absence of an analyte in a clinical specimen including viral, bacterial, fungal associated antigens, tumor markers, cell surface markers, drugs, hormones, and the like derived from clinical specimens such as blood, plasma, serum, cerebrospinal fluid, lymph or tissue fluid, wounds, pus, cells, tissue biopsies, urine or fecal material, swabs or lavages of the urogential, nasopharyngeal, anal, and conjunctival areas of the body.
In addition, as the testing facility is at satellite laboratories, physicians offices, or the home, the existence and performance of a noninstrumented simple assay is desirable, especially where a poorly trained or lay person may perform the test. Noninstrumented immunoassay systems are not capital intensive and can have greater utility and flexibility away from the clinical laboratory. Improvements in simplicitity, speed, specificity, and sensitivity have been ongoing challenges to those individuals involved in developing diagnostic test kits.
The methodologies for noninstrumented qualitative or semi quantitative sandwich immunoassay techniques have included enzyme immunoassays such as those described in 1971 by Engvall and Perlmann in Immunochemistry 8: 871 and Van Weeman and Schuurs in FEBS lett. 15: 232. Further development of sandwhich enzyme immunoassays with chromogenic substrate developed on the surface of a ligand or antiligand coated porous membrane, paper, strip, or film were described by Valkirs et al in U.S. Pat. No. 4,632,901 (1986) and by Frickey et al in U.S. Pat. No. 4,670,381 (1987). Nonenzyme labeled assays on bibulous paper using dyed particles as labels were disclosed by Gould et al in U.S. Pat. No. 4,552,839 (1985). The use of colloidal gold labeled antibodies in an immunoassays were reported by Horrisberger et al in 1977 in Journal of Histochem & Cytochem 25: 295. Horrisberger describes a gold sol immunoassay for mannan wherein there is a change in light absorption upon the aggregation of colloidal gold labeled antibodies. A similar metal sol particle immunoassay is described by Leuvering in U.S. Pat. No. 4,313,734 (1982). In addition Leuvering discloses a sandwich gold sol immunoassay wherein antibody coated plastic plates are coated with specific antibody, followed by the addition of test sample, and incubated. The plate is then washed, gold labeled anti ligand antibody is added, and again incubated. The plate is washed again, and an eluting buffer is added to elute the bound colloidal gold and then read spectrophotometrically. Cerny in patent application #8,502,534 discloses the use of colloidal gold as a label for antibodies in a sandwich assay wherein the bound and unbound labeled antibodies or antigens are separated on an antibody coated porous membrane through radial diffusion. Jolley et al describe a particle concentration fluorescence immunoassay in J. Immunol. Meth (1984) 67: 21 wherein a sandwich assay is performed using antibody coated particles, fluorescent labeled antibodies and a filtration membrane which captures and concentrates the coated particles and bound fluorescent dye which is washed and then measured by front surface fluorometry.
Gefter in U.S. Pat. No. 4,459,361 (1984) discloses a ligand assay with one or two particulate reagents and filter, wherein agglutinated antibody coated dyed particles are separated from nonagglutinated particles by use of a controlled pore size membrane filter. Edwards et al disclose in U.S. Pat. No. 4,666,863 (1987) an immunoassay with chromatographic medium and labeled reagent wherein specific binder for an analyte is attached to solid phase particles. The particles are chosen so that when applied to a spot on a flat sheet chromatographic medium, they do not migrate to any significant extent under the influence of a subsequent applied solvent, while the unbound labeled reagent migrates away from the spot.
The potential of separating bound and unbound labeled materials by attaching one component of reactants to particles and capturing the particles by either diffusion, filtration, capillary action, or chromatography in which the solid phase particles remain immobilized is now well known to those skilled in the art. However, the labels used are of a nature that because of nonspecific interactions, washing steps are required for separation of the bound and unbound label. In addition all of these solid phase particles assays suffer from the following drawbacks required of a simple immunoassay system:
1. There are too many steps for those assays which utilize enzymes, requiring at least one washing step and or addition of a substrate reagent, and the timing of reading is critical.
2. Those assays which require some kind of instrumentation for reading such as in the case of a fluorescent dye are unsuitable for many testing sites.
3. Those assays which require sequential additions of reagents, as in standard enzyme immunoassay procedures, are more difficult to perform, time consuming, and more costly to manufacture multiple reagents.
4. Those assays performed on ligand or antiligand coated porous films, filter, or membranes must be prepared specifically for each particular analyte to be tested and because of limited surface area and reaction time with antigens, this methodology may be inappropriate if the affinity of the antiligand for the ligand is low.